MDS analysis

Snp calling with freebayes

First, need to call SNPs using all BAM files at once:

working in directory 2019/IreneSnps on whitney

first, sort bams

for f in SNPanalysis/*LT*rmdup.bam
    do 
       newname=`basename $f .bam`_sort.bam
       samtools sort -o $newname --reference SNPanalysis/Pinsaporeference1 $f 
    done

next, assign read groups to bams

input=""

for f in `ls *sort.bam`
   do
      rg=`basename $f _rmdup_sort.bam`
      input="$input -b $f -r $rg -s $rg"
  done

echo $input

bamaddrg $input > LT_rmdup_sort_combined.bam

samtools index LT_rmdup_sort_combined.bam

freebayes -f SNPanalysis/Pinsaporeference1 --no-indels --no-mnps --no-complex LT_rmdup_sort_combined.bam > LT.vcf &

try parallel

ulimit -n 4000
/usr/local/stow/freebayes/scripts/fasta_generate_regions.py Pinsaporeference1.fai 100000 > regions
./freebayes-parallel regions 8 -f Pinsaporeference1 --no-indels --no-mnps --no-complex LT_rmdup_sort_combined.bam > LT.vcf 

(note: I edited the freebayes-parallel script so that it would work…)

Freeybayes parallel takes about 12 hours

scp whitney.plb.ucdavis.edu:2019/IreneSnps/LT.vcf.gz ../input/

now analyze vcf in R

library(tidyverse)

get the vcf header

vcf.header <- system("zgrep '#C' ../input/LT.vcf.gz",intern = TRUE) 
vcf.header

[1] "#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\tFORMAT\t38LTR\t42LTR\t42LTRR\t43LTR\t43LTRR\t49LTWR\t49LTWRR\t95LTWR\t95LTWRR\t99LTWR"

vcf.header <- vcf.header %>% 
  str_replace("#","") %>% #get rid of the pound sign
  str_split(pattern = "\t") %>% #split on the tabs
  magrittr::extract2(1)
vcf.header

 [1] "CHROM"   "POS"     "ID"      "REF"     "ALT"     "QUAL"    "FILTER"  "INFO"    "FORMAT"  "38LTR"   "42LTR"   "42LTRR"  "43LTR"   "43LTRR"  "49LTWR"  "49LTWRR"
[17] "95LTWR"  "95LTWRR" "99LTWR" 

get the data

snps <- read_tsv("../input/LT.vcf.gz", na = c("","NA","."),comment="#",col_names = vcf.header) %>%
  select(-ID, -FILTER) # these are empty columns

Parsed with column specification:
cols(
  CHROM = col_character(),
  POS = col_integer(),
  ID = col_character(),
  REF = col_character(),
  ALT = col_character(),
  QUAL = col_double(),
  FILTER = col_character(),
  INFO = col_character(),
  FORMAT = col_character(),
  `38LTR` = col_character(),
  `42LTR` = col_character(),
  `42LTRR` = col_character(),
  `43LTR` = col_character(),
  `43LTRR` = col_character(),
  `49LTWR` = col_character(),
  `49LTWRR` = col_character(),
  `95LTWR` = col_character(),
  `95LTWRR` = col_character(),
  `99LTWR` = col_character()
)

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snps

filter to keep snps where there is data from all samples

snps <- snps %>% 
  filter({select(., matches("[0-9]")) %>% complete.cases() })
snps

snps <- snps %>% 
  filter(QUAL >=50,
         nchar(ALT)==1)
snps